D. melanogaster S2R+ cells were a gift from N. Perrimon’s laboratory (Harvard Medical School, Boston, MA). Human 786-O, 769-P, and A498 cells were originally obtained from the American Type Culture Collection. UMRC-2 cells were originally provided by B. Zbar and M. Linehan (National Cancer Institute, Bethesda, MD) (38). S2R+ cells were maintained in Schneider’s Drosophila Media (Life Technologies, no. 21720024). 786-O, A498, and UMRC-2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, no. 11965126). 769-P cells were maintained in RPMI (Life Technologies, no. 11875119). All media were supplemented with 10% fetal bovine serum (Life Technologies, no. 10437028) and 1× penicillin-streptomycin (Life Technologies, no. 15140163). S2R+ cells were maintained at 25°C and ambient CO2, and all human cells were maintained at 37°C and 5% CO2. S2R+ cells were allowed to grow to confluency and were detached from culture plates by washing with spent media. All human cell lines were passaged at ≤80% confluency using 0.25% trypsin-EDTA (Life Technologies, no. 25200114) to dissociate cells from culture flask. Cells were tested for mycoplasma at least every 8 weeks using the MycoAlert Mycoplasma Detection Kit (Lonza, no. LT07-418).

Where indicated, the following chemicals were added to the media: palbociclib (1 mM stock in water; Selleckchem.com, no. S1116), abemaciclib (10 mM stock in DMSO; a gift from Eli Lilly, no. LY2835219), FG4592 (100 mM stock in DMSO; ApexBio Technology, no. ASP4187), and PT2399 (10 mM stock in DMSO; a gift from Peloton Therapeutics, no. PT2399-16). All stock solutions were stored at −20°C.

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